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Recent progress in molecular biology based upon rapidly developing DNA technology is reviewed. Emphasis is placed on the developing human genome project that includes structural as well as functional analyses of the genome. Expression profiling of active genes in the body helps construct a data-base for the functional aspects of the human genome.
In the first phase of the Human Genome Project, new and ingenious tools have made it possible to map all the individual nucleotides that make up the 23 human chromosomes. During the next 5 years, the 3 billion DNA bases and the 50,000 to 100,000 genes will be sequenced. This knowledge will have widespread applications in biology, medicine and industry. The genetic research community currently has access to abundant DNA markers, detailed chromosome maps, extensive online databases as well as rapid DNA analysis technologies, all of which can be used to identify disease-causing genetic mutations. In the next 15 to 20 years, the Human Genome Project is expected to identify defective genes causing thousands of hereditary diseases, including common diseases such as heart disease, diabetes, asthma and cancer. The hope is that these discoveries will lead to better understanding of the causes of these diseases, and to better approaches to diagnosis, prevention and treatment of human genetic disorders.
Technologies that permit rapid investigation of DNA sequences, such as those containing single nucleotide polymorphisms (SNPs), are of great consequence to many sectors that perform molecular diagnostic analyses. We have developed a novel fluorescent oligonucleotide probe technology, termed HyBeacons, which provides a new homogeneous method for fluorescence-based sequence detection, allele discrimination and DNA quantification. Hybridization of HyBeacons to complementary DNA target sequences results in a measurable elevation of probe fluorescence emission. HyBeacon probes may be incorporated into real-time polymerase chain reaction (PCR) assays to detect the presence and monitor the accumulation of specific DNA sequences. Furthermore, closely related sequences differing by as little as a single nucleotide may be discriminated by measuring the melting temperatures (T(m)) of various probe/target duplexes and exploiting the differences in T(m) that exist between different duplexes. We demonstrate here that HyBeacon probes are efficient tools for rapid sequence analysis and that a single probe may be employed to reliably identify homozygous and heterozygous samples. Additional benefits exhibited by the HyBeacon technology derive from their simple mode of action, ease of design, relatively inexpensive synthesis and potential for multiplex analysis.
Current sequencing technologies are insufficient to cope with large-scale projects such as sequencing the human genome and genomes of model organisms. In addition, as genetic lesions associated with specific human diseases are identified, DNA sequencing will be used increasingly for clinical applications. Thus, new approaches are needed to combine high-throughput with accuracy for both research and diagnostic purposes. A novel technology based on detection of individual fluorescent nucleotides in a flowing sample stream is under development.
Rapid and continuous progress in biotechnology is leading industry to increasing application of recombinant DNA (rDNA) technology in the production of drug substances. The main motives are obtaining products in larger amounts and better quality by means of comparably economical methods. For reasons of safety and purity some national regulatory agencies are excluding drug substances of natural origin, e.g. human growth hormone isolated from pituitary glands, where the same products obtained by rDNA are available. General and specific literature on biotechnology is widely available but official guidelines and regulatory requirements for registration, manufacture and control of pharmaceuticals produced by rDNA are still exceptional. In Switzerland the experience in the field of rDNA-based large scale production of pharmaceuticals is limited. The registration of the preparations and the inspection of their industrial production therefore take place on a case by case basis. For that purpose the relevant "Points to consider" issued by the American Food and Drug Administration (FDA) are essential. A comparison between conventional manufacture and rDNA technology is made by indicating some examples.
Allele-specific oligonucleotide probes, together with dot-blot methods, can provide rapid and inexpensive screening of DNA types in large samples of organisms. Here we demonstrate their use in: (1) determining types of mitochondrial DNA in hundreds of lizards from a dynamic hybrid zone; (2) discovering intraspecific geographic variation in genes; and (3) determining and verifying the maternal ancestry of unisexual, parthenogenetic lizards in clones of hybrid origin. These methods are broadly applicable in research involving rapid screening of DNAtypes in large samples of specimens for any gene with sequence data from which to design specific probes.
Two systems, biphasic MB-Check and radiometric BACTEC, based on liquid media proved to be significantly better than the egg-based solid media for the isolation of mycobacteria from clinical specimens. The difference in the rates of isolation of mycobacteria between two groups of media was more remarkable with smear-negative specimens. The time to the detection of the Mycobacterium tuberculosis complex with MB-Check was shorter than that with the 3% Ogawa egg method but longer than that with BACTEC. A total of 135 sputum specimens were examined by the polymerase chain reaction (PCR) using oligonucleotides based on the repetitive sequence (IS986) of M. tuberculosis as a primer. The PCR gave an overall positivity rate of 84.2%, as compared with 71.9% by smear and 96.9% by culture in the liquid medium, MB-Check. Although the sensitivity of the PCR appeared to be similar to that of culture with the MB-Check system, the PCR should be very useful for rapid detection of M. tuberculosis infections. DNA probe technology facilitates a rapid and specific identification of microorganisms. The diagnostic specificity and sensitivity of the nonradioactive Accuprobe M. tuberculosis complex and Accuprobe M. avium complex culture confirmation tests were nearly 100%. The results of the colorimetric microdilution plate hybridization test (DDH-Mycobacteria) for identification of mycobacteria were consistent with those of biochemical identification. About 90% of clinical isolates could be identified by the DDH kit. Both of these methods may contribute to the rapid diagnosis of mycobacterial infections. Epidemiological studies with techniques which allow differentiation of strains within M. tuberculosis groups are important for limiting the dissemination of the disease.(ABSTRACT TRUNCATED AT 250 WORDS)
Current methods for the identification of Mycobacterium tuberculosis are dependent upon culture of the bacteria and are necessarily lengthy due to the slow growth of this agent. The development of DNA probe technology offers rapid, accurate and cost effective alternatives for the identification of such fastidious organisms. A technique for detecting specific DNA sequences, known as oligonucleotide ligation assay (OLA) involves the ligation of two adjacent oligonucleotides annealed to target DNA, and has been previously described. Amplification of the target sequences can be accomplished by including complementary pairs of oligonucleotides and a thermal stable ligase in a reaction which cycles between annealing/ligation and denaturing temperatures. Using a cloned portion of an insertion sequence, IS6110, which has been reported to be specific for M. tuberculosis complex as target DNA, we demonstrate the ligation dependent amplification of a 40 base pair region of plasmid bearing IS6110. By employing oligonucleotides which are each labelled with a different fluorescent dye, the reaction can be followed by fluorescence detection on an Applied Biosystems model 373A DNA sequencer. Using this approach, we have optimized conditions for the detection of 100 target molecules in a mixture containing 4 micrograms of unrelated DNA. Since the insertion sequence is repeated on average 12-14 times in the genome of M. tuberculosis, this corresponds to a theoretical detection level of 7-8 organisms. Completion of this entire assay can be accomplished in less than 8 h and serves as a basis for further studies in the development of a rapid clinical diagnostic test for tuberculosis.
Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici, and Verticillium wilt, caused by either Verticillium albo-atrum or V. dahliae, are devastating diseases of tomato (Lycopersicon esculentum Mill.) found worldwide. Monitoring is the cornerstone of integrated pest management of any disease. The lack of rapid, accurate, and reliable means by which plant pathogens can be detected and identified is one of the main limitations in integrated disease management. In this paper, we describe the development of a molecular detection system, based on DNA array technology, for rapid and efficient detection of these vascular wilt pathogens. We demonstrate that by using this array these pathogens can be detected within 24 h from complex substrates like soil, plant material, and samples as they are collected by tomato growers in their greenhouses.
Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici, and Verticillium wilt, caused by either Verticillium albo-atrum or Verticillium dahliae, are devastating diseases of tomato (Lycopersicon esculentum) found worldwide. Monitoring is the cornerstone of integrated pest management of any disease. The lack of rapid, accurate, and reliable means by which plant pathogens can be detected and identified is one of the main limitations in integrated disease management. In this paper, we describe the development of a molecular detection system, based on DNA array technology, for rapid and efficient detection of these vascular wilt pathogens. We show the utility of this array for the sensitive detection of these pathogens from complex substrates like soil, plant tissues and irrigation water, and samples that are collected by tomato growers in their greenhouses.
DNA microchip technology is a rapid, high-throughput method for nucleic acid hybridization reactions. This technology requires random fragmentation and fluorescent labeling of target nucleic acids prior to hybridization. Radical-generating coordination complexes, such as 1,10-phenanthroline-Cu(II) (OP-Cu) and Fe(II)-EDTA (Fe-EDTA), have been commonly used as sequence nonspecific "chemical nucleases" to introduce single-strand breaks in nucleic acids. Here we describe a new method based on these radical-generating complexes for random fragmentation and labeling of both single- and double-stranded forms of RNA and DNA. Nucleic acids labeled with the OP-Cu and the Fe-EDTA protocols revealed high hybridization specificity in hybridization with DNA microchips containing oligonucleotide probes selected for identification of 16S rRNA sequences of the Bacillus group microorganisms. We also demonstrated cDNA- and cRNA-labeling and fragmentation with this method. Both the OP-Cu and Fe-EDTA fragmentation and labeling procedures are quick and inexpensive compared to other commonly used methods. A column-based version of the described method does not require centrifugation and therefore is promising for the automation of sample preparations in DNA microchip technology as well as in other nucleic acid hybridization studies.
In order to investigate allele loss in colorectal tumours we have developed a rapid technique which overcomes most of the problems associated with radioactive Restriction Fragment Length Polymorphism (RFLP) analysis of allele loss. We utilise microsatellite length polymorphisms which are highly informative and are closely linked to loci of interest. Sequences containing microsatellites can be amplified from normal and tumour DNA pairs by a polymerase chain reaction (PCR) in which one of the primers is fluorescently labelled. This enables us to detect the products on polyacrylamide gels run on an automated DNA sequencer using dedicated software, by which results are automatically quantitated in terms of peak size, height, and area. Using this technique we have analysed 26 normal tissue: cancer pairs for allele loss at two loci linked to the adenomatous polyposis coli (APC) gene on chromosome 5q. Repeated assays yielded identical results for each pair. Allele loss was found in 10 out of 25 informative samples (40%).
The ability to rapidly recognize severe acute respiratory syndrome coronavirus (SARS-CoV) as a cause of infections is critical to quickly limiting further spread of the disease. A rapid, sensitive, and specific laboratory diagnostic test is needed to confirm outbreaks of SARS-CoV infection and distinguish it from other diseases that can cause similar clinical symptoms. An improved TaqMan technology using minor groove binder (MGB) probes was used to detect and quantify SARS-CoV in suspected patients. SARS-CoV primers and probe were designed based on the open reading frame 1b sequence, which encodes coronavirus replicase protein. A linear standard curve with R2 > 0.99 was obtained, and the threshold sensitivity was 10 genome equivalents per reaction. Interassay coefficients of variation were 1.73 to 2.72%, indicating good reproducibility of this method. A total of 228 specimens from 151 suspected patients were quantified by this method, 13 specimens from 6 patients were positive with viral load range from 362 to 36,240,000 genome equivalents/mL. In conclusion, the high sensitivity and reproducibility of the real-time polymerase chain reaction SARS-CoV RNA quantitation using MGB probe allowed the screening of large numbers of clinical samples.
Rapid progress in DNA technology has entailed the possibility of readily detecting mutations in disease genes. In contrast to this, techniques to characterize the effects of mutations are still very time consuming. It has turned out that many of the mutations detected in disease genes are missense mutations. Characterization of the effect of these mutations is particularly important in order to establish that they are disease causing and to estimate their severity. We use the experiences with investigation of medium-chain acyl-CoA dehydrogenase deficiency as an example to illustrate that (i) impaired folding is a common effect of missense mutations occurring in genetic diseases, (ii) increasing the level of available chaperones may augment the level of functional mutant protein in vivo, and (iii) one mutation may have multiple effects. The interplay between the chaperones assisting folding and proteases that attack folding intermediates is decisive for how large a proportion of a mutant polypeptide impaired in folding acquires the functional structure. This constitutes a protein quality control system, and the handling of a given mutant protein by this system may vary due to environmental conditions or genetic variability in its components. The possibility that intraindividual differences in the handling of mutant proteins may be a mechanism accounting for phenotypic variability is discussed.
Since the discovery of the first oncogene 26 years ago, a large body of research has convincingly demonstrated that the initiation and progression of cancers involve the accumulation of genetic aberrations in the cell. Many techniques have been developed to identify these genetic abnormalities. The recent completion of human genome sequencing and advances in DNA microarray technology allow rapid genetic analysis to take place on a genome-wide scale and have revolutionized the way cancers are studied. This ground-breaking approach of studying cancer promises to provide a better understanding of the underlying mechanism for tumorigenesis, more accurate diagnosis, more comprehensive prognosis, and more effective therapeutic interventions.
SETTING: The rapid detection of Mycobacterium tuberculosis (TB) in clinical samples is an important goal. The LightCycler heralds an advance in thermal cycle technology combining rapid cycle DNA amplification with fluorimetry, eliminating the need to perform amplification and product analysis separately. OBJECTIVES: To evaluate the LightCycler for direct detection of M. tuberculosis complex in respiratory specimens. To evaluate a DNA extraction method based on Chelex 100 resin, heating and ultrasonication for the prevention of endogenous inhibitions in respiratory samples. DESIGN: DNA was extracted from sputum samples using the Chelex method and polymerase chain reaction (PCR) for TB performed with the LightCycler. RESULTS: For 88 sputum samples positive by microscopy and culture for M. tuberculosis, 95% were PCR-positive. None of the five sputum samples that were smear-negative but culture-positive for M. tuberculosis, the 79 culture-negative sputum samples and the 29 sputum samples that were culture-positive for mycobacteria other than TB yielded positive PCR results. PCR inhibitors were not detected in any of the samples. CONCLUSION: The LightCycler proved a simple, reproducible and rapid system, reducing the time to result from weeks (culture) or days (conventional PCR) to hours. The Chelex 100 resin method produced good results for the smear-positive specimens. However, a larger study is required to determine the efficacy of the method with smear-negative specimens and for specimens known to contain endogenous inhibitors.
The ability to diagnose human diseases at the DNA level has become possible because of a rapid development in DNA technology, particularly in the area of detection of single base substitutions. Mutations in the genomic DNA of a particular gene may be inferred indirectly using linkage analysis and restriction fragment length polymorphisms. However, direct detection of the mutation is the more favourable approach. The advent of the polymerase chain reaction to amplify specific regions of genomic DNA or mRNA has enhanced the speed and sensitivity of many of the screening and diagnostic procedures. Screening methods have been developed that will detect at least 70% and, with some methods, close to 100% of all mutations. The methods include ribonuclease A cleavage, denaturing gradient gel electrophoresis, chemical cleavage of mismatch and direct sequencing. Choice of method is based on a number of factors and will depend on the structure of the gene to be analysed. Following identification of a mutation using one of the screening procedures, prenatal diagnosis and carrier testing can be offered. The overall aim is to develop a method that has the potential to determine the mutation present in an index case of a previously untested family in a few days, thus allowing any other relevant family member to be tested.
Molecular characterization of allergens by recombinant DNA technology has made rapid progress in the recent few years. In the present study we immunized mice with aluminum hydroxide-adsorbed purified recombinant major timothy grass pollen allergens (rPhl p 1, rPhl p 2, rPhl p 5), dog albumin, a major animal dander allergen, and proteins with low (beta-lactoglobulin) or no (ribulose diphosphate carboxylase) allergenic potential in humans. Allergens that bind high levels of IgE in humans (Phl p 1, Phl p 5, dog albumin) induced high IgE and IgG1 levels in mice, whereas proteins with little or no allergenic activity in humans failed to induce significant IgE and IgG1 levels in mice. Continuous immunization for a period of 27 wk resulted in the production of mouse IgG1 Abs that recognized recombinant allergen fragments/epitopes defined by IgE Abs of allergic patients. As a consequence, allergen-specific mouse Abs strongly inhibited human IgE binding to the allergens and suppressed the allergen-induced histamine release from human basophils. In summary, our data indicate that 1) the allergenic potency of a protein may be related to its overall immunogenicity and 2) prolonged immunization with single purified recombinant allergens induces protective IgG Abs. The presented experimental in vivo/in vitro system allows the evaluation of Ag preparations (e.g., recombinant allergens) to be used for immunotherapy in humans.